Project summary Despite effective antiretroviral therapy (ART), HIV-1 persists in memory CD4+ T cells as the major barrier to cure. It was recently proposed that HIV-1 can drive the aberrant proliferation of the infected cells through integration into cancer-related genes. The clonally expanding latent reservoir, if present, hampers HIV-1 eradication efforts and should be targeted specifically. However, it remains unclear whether HIV-1 proviruses integrated into cancer-related genes are intact or defective, and how HIV-1 may drive the clonal expansion through integration into cancer-related genes. Several challenges prevent the study of HIV-1 persistence. First, the rarity of HIV-1 infected cells and the lack of reliable markers which can distinguish cells containing inducible HIV-1 (~1-10 per million resting CD4+ T cells) from cells containing defective HIV-1 (~100-1000 per million resting CD4+ T cells) and uninfected cells makes HIV-1-specific analysis difficult. Second, transcriptome analysis of bulk CD4+ T cells from HIV-1-infected individuals captures mostly the transcriptome of HIV-1 uninfected cells. Third, methods studying HIV-1 integration sites disrupt the HIV-1 genome, while methods studying HIV-1 full-length sequences and replication competence exclude HIV-1 integration sites from amplification. To this end, we developed the innovative, cutting-edge HIV-1 RNA SortSeq which identifies cells containing inducible HIV-1 for single cell RNAseq analysis and the integration site of inducible HIV-1. From blood samples obtained from virally suppressed individuals, we identified HIV-1-host chimeric RNA which depicts inducible HIV-1 RNA and HIV-1 integration sites at the same time. Further, we identified three patterns of HIV-1-host interactions: 1) read-through transcription, 2) host RNA splicing into HIV-1 RNA, creating novel transcription variants encoding a host-HIV-1 fusion protein, and 3) HIV-1 RNA splicing into host RNA, indicating HIV-1 driven host (cancer- related) gene expression. We hypothesize that HIV-1 which are integrated into cancer-related genes may drive the proliferation of the infected cells and promote HIV-1 persistence through HIV-1-host RNA interactions. Our goal is to examine whether HIV-1 integration into cancer-related genes causes clonal expansion (Aim 1) and to identify the mechanisms of HIV-1-driven proliferation (Aim 2). In Aim 1, we will obtain blood samples from virally suppressed individuals at different time points to determine whether inducible HIV-1 which are integrated into cancer-related genes undergo clonal expansion using HIV-1 RNASortSeq. We will determine whether HIV-1 integration into cancer-related gene changes the host cell transcriptome at the single cell level. We will examine the contribution of T cell activation, antigen-driven proliferation and homeostatic proliferation in HIV-1 clonal expansion. In Aim 2, we will use our established cell line model to examine HIV-1- host RNA interactions and clonal expansion upon HIV-1-specific and integration site-specific stimulations. Overall, we will examine HIV-1 persistence and clonal expansion at the single cell level, which will facilitate the development of a more effective HIV-1 cure strategies targeting the clonally expanding latent reservoir.